We propose to continue our studies on the molecular properties and the sarcomeric organization of two extremely large, major myofibrillar proteins\-- titin and nebulin -- which are found in the skeletal and cardiac muscles of a wide range of vertebrate and invertebrate species. Our studies in the past years have indicated that titin and nebulin may be components of an elastic, continuous myofilament (the "third" filament) -- distinct from this and thick filaments -- within the sarcomere. In the next grant period, we propose to (a) investigate further the molecular properties of titin and nebulin emphasizing the purification of native proteins, the study of domain organization, the study of immunological properties, the visualization of morphology and the investigation of their interactions with other myofibrillar proteins; (b) continue and extend our antibody localization studies to include the use of monoclonal antibodies and electron microscopic techniques, emphasizing the detection and labeling of ultrathin filaments in the sarcomere; (c) characterize the phosphorylation and calcium binding properties of titin and nebulin, emphasizing the possible involvement of these proteins in regulatory mechanisms; and (d) perform a survey of the distribution and organization of titin and nebulin in nature, emphasizing their presence in insect flight muscle and in nonmuscle cells. We hope that the biochemical and structural studies proposed here will lead to better understanding of the structure and function of contractile machinery, the mechanical properties of striated muscle and the mechanism of muscle contraction and relaxation.